B cells are a critical component of the immune response in mammals, as they are the cells responsible for antibody production (humoral immunity). Each B cell within the host expresses a different antibody—thus, one B cell will express antibody specific for one antigen, while another B cell will express antibody specific for a different antigen. Accordingly, B cells are quite diverse, and this diversity is critical to the immune system. In humans, each B cell can produce an enormous number of antibody molecules (i.e., about 107 to 108). The maturation of B cells (and thus antibody production) most typically ceases or substantially decreases when the foreign antigen has been neutralized. Occasionally, however, proliferation of a particular B cell or plasma cell will continue unabated; such proliferation can result in a cancer referred to as “B cell lymphoma or multiple myeloma.”
B cell lymphomas include both Hodgkin's lymphoma and a broad class of non-Hodgkin's lymphoma. Non-Hodgkin's lymphoma encompasses over 29 types of lymphoma. The distinctions are based on the type of cancer cells, and often the cancers are classified by cell type and rate of growth.
The incidence of non-Hodgkin's lymphoma is much greater than the incidence of Hodgkin's lymphoma: about 8 in 9 cases. According to the American Cancer Society, an estimated 54,000 new non-Hodgkin's lymphoma cases will be diagnosed, 65% of which will be classified as intermediate- or high-grade lymphoma. Patients diagnosed with intermediate-grade lymphoma have an average survival rate of two to five years, and patients diagnosed with high-grade lymphoma survive an average of six months to two years after diagnosis.
Diffuse large B cell lymphoma (DLBCL) is an aggressive and the most common subtype of non-Hodgkin's lymphoma, accounting for 30-40% of lymphoid malignancy. Although DLBCL represents one of the most therapy-responsive malignancies, only approximately 40% of the patients can be cured by the current treatments with multiple therapeutic agents (Abramson et al., “Advances in the Biology and Therapy of Diffuse Large B-cell Lymphoma: Moving Toward a Molecularly Targeted Approach,” Blood 106:1164-1174 (2005); Staudt et al., “The Biology of Human Lymphoid Malignancies Revealed by Gene Expression Profiling,” Adv Immunol 87:163-208 (2005)). As a result, nearly 10,000 patients die from DLBCL each year in the United States, (Staudt et al., “The Biology of Human Lymphoid Malignancies Revealed by Gene Expression Profiling,” Adv Immunol 87:163-208 (2005)), underscoring the importance of better molecular understanding of DLBCL and identifying proper therapeutic targets. Based on gene expression profiles, DLBCL can be divided into at least three subgroups: activated B cell-like (ABC) DLBCL, germinal center B cell-like (GCB) DLBCL, and primary mediastinal B cell lymphoma (PMBL), with the ABC and PMBL subgroups exhibiting higher levels of expression of NF-κB target genes than the GCB subgroup (Rosenwald et al., “Molecular Diagnosis of Primary Mediastinal B Cell Lymphoma Identifies a Clinically Favorable Subgroup of Diffuse Large B Cell Lymphoma Related to Hodgkin Lymphoma,” J Exp Med 198:851-862 (2003); Wright et al., “A Gene Expression-based Method to Diagnose Clinically Distinct Subgroups of Diffuse Large B Cell Lymphoma,” Proc Natl Acad Sci USA 100:9991-9996 (2003); Alizadeh et al., “Distinct Types of Diffuse Large B-cell Lymphoma Identified by Gene Expression Profiling,” Nature 403:503-511 (2000); Savage et al., “The Molecular Signature of Mediastinal Large B-cell Lymphoma Differs from That of Other Diffuse Large B-cell Lymphomas and Shares Features with Classical Hodgkin Lymphoma,” Blood 102:3871-3879 (2003)). It was shown that constitutive NF-κB signaling is required for survival of ABC and PMBL DLBCL cells (Feuerhake et al., “NFκB Activity, Function, and Target-gene Signatures in Primary Mediastinal Large B-cell Lymphoma and Diffuse Large B-cell Lymphoma Subtypes,” Blood 106:1392-1399 (2005); Davis et al., “Constitutive Nuclear Factor κB Activity is Required for Survival of Activated B Cell-like Diffuse Large B Cell Lymphoma Cells,” J Exp Med 194:1861-1874 (2001)), and that small molecules that inhibit IκB kinases (IKK) are selectively toxic for these two DLBCL subgroup cells (Lam et al., “Small Molecule Inhibitors of IκB Kinase Are Selectively Toxic for Subgroups of Diffuse Large B-cell Lymphoma Defined by Gene Expression Profiling,” Clin Cancer Res 11:28-40 (2005)). These studies highlight the NF-κB pathway as a promising therapeutic target in B-lymphomas that depend on NF-κB activity for proliferation and survival.
Multiple myeloma (“MM”) is a B cell malignancy characterized by the latent accumulation in bone marrow of secretory plasma cells with a low proliferative index and an extended life span. The disease ultimately attacks bones and bone marrow, resulting in multiple tumors and lesions throughout the skeletal system. Approximately 1% of all cancers, and slightly more than 10% of all hematologic malignancies, can be attributed to multiple myeloma. Incidence of MM increases in the aging population, with the median age at time of diagnosis being about 61 years. Current treatment protocols, which include a combination of chemotherapeutic agents such as vincristine, β-chloro-nitrosourea (BCNU), melphalan, cyclophosphamide, Adriamycin, and prednisone or dexamethasone, yield a complete remission rate of only about 5%, and median survival is approximately 36-48 months from the time of diagnosis. Recent advances using high dose chemotherapy followed by autologous bone marrow or peripheral blood progenitor cell (PBMC) transplantation have increased the complete remission rate and remission duration. Yet overall survival has only been slightly prolonged, and no evidence for a cure has been obtained. Ultimately, all MM patients relapse, even under maintenance therapy with interferon-α (IFN-α) alone or in combination with steroids.
MM has been previously associated with enhanced NF-κB activity (Berenson et al., “The Role of Nuclear Factor-κB in the Biology and Treatment of Multiple Myeloma,” Semin. Oncol. 28(6):626-33 (2001). New evidence on the molecular mechanisms that underlie aberrant NF-κB activity in MM tumor cells has been found recently (Annunziata et al., “Frequent Engagement of the Classical and Alternative NF-κB Pathways by Diverse Genetic Abnormalities in Multiple Myeloma,” Cancer Cell 12(2):115-30 (2007); Keats et al., “Promiscuous Mutations Activate the Noncanonical NF-κB Pathway in Multiple Myeloma,” Cancer Cell 12(2):131-44 (2007)). The results reported in these studies confirm that diverse mutations can lead to pathological activation of NF-κB signaling in MM, and result in a shift of plasma cells from dependence on the microenvironment to an environment-independent state during progression of MM.
The NF-κB family consists of five members in mammals: p65 (RelA), RelB, c-Rel, NF-κB1(p105/p50), and NF-κB2(p100/p52). Members of NF-κB proteins form homo- or heterodimers and are retained in the cytoplasm prior to activation by the associated IκB proteins, or the precursor proteins p100 or p105, which contain the ankyrin repeats present also in the IκB proteins and, thus, can also function as IκB proteins. Extracellular signals induce NF-κB activation through two major pathways: the classical (also called the canonical pathway) and the alternative (the non-canonical) pathways. Activation of these pathways leads to activation of IKK and degradation or processing of IκB proteins, resulting in the release of sequestered NF-κB proteins and their subsequent translocation into the nucleus and target gene activation (Hayden et al., “Signaling to NF-κB,” Genes Dev 18:2195-2224 (2004); Karin, “NF-κB and Cancer: Mechanisms and Targets,” Mol Carcinog 45:355-361 (2006); Scheidereit, “IκB Kinase Complexes: Gateways to NF-κB Activation and Transcription,” Oncogene 25:6685-6705 (2006)). Abnormal activation of NF-κB contributes to tumor development and progression, as well as to the resistance of cancer cells to chemotherapeutic agents and radiation therapy (Kim et al., “NF-κB and IKK as Therapeutic Targets in Cancer,” Cell Death Differ 13(5):738-47 (2006); Karin, “Nuclear Factor-κB in Cancer Development and Progression,” Nature 441:431-436 (2006)). Thus, inhibition of NF-κB activation represents a promising approach for cancer therapy (Karin et al., “The IKK NF-κB System: A Treasure Trove for Drug Development,” Nat Rev Drug Discov 3:17-26 (2004)). However, as NF-κB is expressed ubiquitously and is involved in a wide variety of normal cellular functions, a general inhibition of NF-κB activity would likely cause serious side effects (Kim et al., “NF-κB and IKK as Therapeutic Targets in Cancer,” Cell Death Differ 13(5):738-47 (2006); Luo et al., “IKK/NF-κB Signaling: Balancing Life and Death—A New Approach to Cancer Therapy,” J Clin Invest 115:2625-2632 (2005); Li et al., “Inflammation-associated Cancer: NF-κB is the Lynchpin,” Trends Immunol 26:318-325 (2005)). To achieve the specificity required for effective therapeutic intervention, it may be necessary to target cancer cell- or signal-specific regulators of NF-κB activation pathways.
The B cell-activating factor belonging to the tumor necrosis factor family (BAFF, also known as BlyS, TALL-1, THANK, zTNF-4, CD257 and TNFSF-13B) is critical for the development and survival of normal B lymphocytes, and activates NF-κB through both the classical and the alternative activation pathways in B cells (Kayagaki et al., “BAFF/BLyS Receptor 3 Binds the B Cell Survival Factor BAFF Ligand Through a Discrete Surface Loop and Promotes Processing of NF-κB2,” Immunity 17:515-524 (2002); Claudio et al., “BAFF-induced NEMO-independent Processing of NF-κB2 in Maturing B cells,” Nat Immunol 3:958-965 (2002); Schneider, P., “The Role of APRIL and BAFF in Lymphocyte Activation,” Curr Opin Immunol 17:282-289 (2005); Sutherland et al., “Targeting BAFF: Immunomodulation for Autoimmune Diseases and Lymphomas,” Pharmacol Ther 112:774-786 (2006); Hatada et al., “NF-κB1 p50 is Required for BLyS Attenuation of Apoptosis but Dispensable for Processing of NF-κB2 p100 to p52 in Quiescent Mature B Cells,” J Immunol 171:761-768 (2003)). Dysregulated BAFF signaling has been associated with various B-cell malignancies. Elevated levels of BAFF have been detected in the serum of patients with various types of B-cell malignancies, and malignant B cells from patients express abnormally high levels of BAFF and one or more BAFF receptors (He et al., “Lymphoma B Cells Evade Apoptosis Through the TNF Family Members BAFF/BLyS and APRIL,” J Immunol 172:3268-3279 (2004); Novak et al., “Expression of BLyS and its Receptors in B-cell non-Hodgkin Lymphoma: Correlation with Disease Activity and Patient Outcome,” Blood 104:2247-2253 (2004); Novak et al., “Aberrant Expression of B-lymphocyte Stimulator by B Chronic Lymphocytic Leukemia Cells: A Mechanism for Survival,” Blood 100:2973-2979 (2002); Briones et al., “BLyS and BLyS Receptor Expression in non-Hodgkin's Lymphoma,” Exp Hematol 30:135-141 (2002); Fu et al., “Constitutive NF-κB and NFAT Activation Leads to Stimulation of the BLyS Survival Pathway in Aggressive B Cell Lymphomas,” Blood 107(11):4540-4548 (2006); Moreaux et al., “BAFF and APRIL Protect Myeloma Cells from Apoptosis Induced by Interleukin 6 Deprivation and Dexamethasone,” Blood 103:3148-3157 (2004); Elsawa et al., “B-lymphocyte stimulator (BLyS) Stimulates Immunoglobulin Production and Malignant B-cell Growth in Waldenstrom's Macroglobulinemia,” Blood 107(7):2882-2888 (2006); Kern et al., “Involvement of BAFF and APRIL in the Resistance to Apoptosis of B-CLL Through an Autocrine Pathway,” Blood 103:679-688 (2004). It has been shown that BAFF functions as a crucial autocrine and paracrine survival factor for malignant B cells (Sutherland et al., “Targeting BAFF: Immunomodulation for Autoimmune Diseases and Lymphomas,” Pharmacol Ther 112:774-786 (2006); He et al., “Lymphoma B Cells Evade Apoptosis Through the TNF Family Members BAFF/BLyS and APRIL,” J Immunol 172:3268-3279 (2004); Novak et al., “Aberrant Expression of B-lymphocyte Stimulator by B Chronic Lymphocytic Leukemia Cells: A Mechanism for Survival,” Blood 100:2973-2979 (2002); Fu et al., “Constitutive NF-κB and NFAT Activation Leads to Stimulation of the BLyS Survival Pathway in Aggressive B Cell Lymphomas Blood 107(11):4540-4548 (2006); Moreaux et al., “BAFF and APRIL Protect Myeloma Cells from Apoptosis Induced by Interleukin 6 Deprivation and Dexamethasone,” Blood 103:3148-3157 (2004); Novak et al., “Expression of BCMA, TACI, and BAFF-R in Multiple Myeloma: A Mechanism for Growth and Survival,” Blood 103:689-694 (2004); Elsawa et al., “B-lymphocyte Stimulator (BLyS) Stimulates Immunoglobulin Production and Malignant B-cell Growth in Waldenstrom's Macroglobulinemia,” Blood 107(7):2882-2888 (2006); Kern et al., “Involvement of BAFF and APRIL in the Resistance to Apoptosis of B-CLL Through an Autocrine Pathway,” Blood 103:679-688 (2004); Endo et al., “BAFF and APRIL Support Chronic Lymphocytic Leukemia B-cell Survival Through Activation of the Canonical NF-κB pathway,” Blood 109:703-710 (2007); Nishio et al., “Nurselike Cells Express BAFF and APRIL, Which Can Promote Survival of Chronic Lymphocytic Leukemia Cells Via a Paracrine Pathway Distinct from That of SDF-1α,” Blood 106:1012-1020 (2005), making the BAFF signaling pathway an attractive therapeutic target for B-cell malignancies. Despite the importance of BAFF signaling in both normal and malignant B cells, the mechanisms by which BAFF activates downstream events, including NF-κB activation, remain to be elucidated.
Protein kinase C-associated kinase PKK (also known as DIK and RIP4) was initially identified as a protein kinase C β- and δ-interacting protein (Bahr et al., “DIK, a Novel Protein Kinase that Interacts with Protein Kinase Cdelta. Cloning, Characterization, and Gene Analysis,” J Biol Chem 36350-36357 (2000); Chen et al., “Protein Kinase C-associated Kinase (PKK), a Novel Membrane-associated, Ankyrin Repeat-containing Protein Kinase,” J Biol Chem 276:21737-21744 (2001)). It belongs to the RIP kinase family, and shares high sequence homology at the N-terminal kinase domain with other members of this kinase family but contains unique C-terminal ankryin repeats (Meylan et al., “The RIP Kinases: Crucial Integrators of Cellular Stress,” Trends Biochem Sci 30:151-159 (2005)). Mice deficient in PKK die soon after birth, likely due to suffocation caused by abnormal epidermal differentiation (Holland et al., “RIP4 is an Ankyrin Repeat-containing Kinase Essential for Keratinocyte Differentiation,” Curr Biol 12:1424-1428 (2002)). It has been shown that PKK activates NF-κB when overexpressed in non-lymphoid cells (Moran et al., “Protein Kinase C-associated Kinase can Activate NFκB in Both a Kinase-dependent and a Kinase-independent Manner,” J Biol Chem 278:21526-21533 (2003); Meylan et al., “RIP4 (DIK/PKK), a Novel Member of the RIP Kinase Family, Activates NF-κB and is Processed During Apoptosis,” EMBO Rep 3:1201-1208 (2002); Muto et al., “Protein Kinase C-associated Kinase (PKK) Mediates Bcl10-independent NF-κB Activation Induced by Phorbol Ester,” J Biol Chem 277:31871-31876 (2002)).
Despite these prior reports on the relationship between PKK and NF-κB activation, these prior reports fail to demonstrate any function of PKK in the proliferation/survival of cancer cells, particularly B cell malignancies such as lymphomas and multiple myeloma.
The present invention is directed to overcoming these and other deficiencies in the art.